The arterial wall and arterial valves are complex macromolecular structures. One of the major elements of these structures is the scaffold that provides the strength and flexibility to perform the task in hand either retaining the blood in vessels against the arterial pressure or maintaining pressure via the function of coronary valves. In the last several years it has become apparent that the actual microstructure and composition of these macromolecules could influence the progress of different disease states most notably atherosclerosis and valve calcification. To gain a better understanding of this process, we have embarked on studies to understand the fine structure of the macromolecules in arterial vascular bed using a novel optical imaging technique that relies on the non-linear excitation (NLE) of collagen and elastin to provide sub-micron images of their structure in unfixed fresh samples together with direct monitoring of water permeation through the wall using Coherent anti-Stokes Raman Scattering(CARS). Using these approaches we have made the following observations: 1) The primary results from this study were published this year (Direct visualization of the arterial wall water permeability barrier using CARS microscopy: see bibliography). 2) One question raised in the review of this manuscript was to attempt these studies on vascular endothelial cells in culture where more control and better optics could be realized. We are currently working with Dr. Huang at Cambridge University to attempt to develop an in vitro cultured vascular endothelial cell preparation with appropriate distribution of proteins in the apical and basolateral membranes. 3) Together with Dr. Dora at the University of Oxford we are expanding our studies on perfused arteries to three systems: A) The rat cremaster artery which physiologically retains it diameter over a wide range pressures, this system may avoid the expansion artifact with deuterium in the lumen of the vessel. This could greatly improve the spatial resolution in determining the water gradients across the wall. B) The mouse internal cerebral arteries, naturally the water handling in this volume restricted structure, the skull, is of interest. In addition, it has been reported the literature that AQP-1 is not present in small arteries but AQP-4 is. We intend on confirming this observation and see if AQP-4 is similarly localized as we found in the mesenteric artery and how this AQP-4 might be regulated in the brain. C) We are re-establishing an AQP-1 knockout mouse to test our hypothesis that the apical membrane is highly permeable to water due the presence of AQP-1.